There have been numerous apparatus and methods for the determination of concentrations in samples in the past. Methods which employ reactions to form colored complexes and analysis of molecules which have an inherent ability to absorb light are governed by Beer's Law. Beer's Law states that at a specified wavelength of light the concentration of an analyte is directly proportional to the amount of light energy absorbed at a constant path length of light. The extent of light absorption and subsequently the analyte concentration is calculated from data generated by an electro-optical device called a colorimeter.
A colorimeter employs a light source, a method for selecting specific light energy, a device with a fixed optical path length for retention of the colored complex or molecules to be quantified and a detection system. The detection system consists of a device to measure light intensity and appropriate electronic circuitry to prevent the results in the desired format. The more precise the measurement required for calculating the concentration, the more sophisticated the design of the colorimeter must be. Most often data are presented as absorbance which is calculated by taking the logarithm (base 10) of the quantity, intensity of light incident to the sample divided by intensity of light transmitted through the sample.
When the absorption of light energy by a sample is large, which is the case with a high concentration of analyte, a reliable value cannot be determined because the detected signal is outside of the operating range of the colorimeter. In this case, the chemistry of the analysis may continue to obey Beer's Law, but instrument limitations prevent the accurate measurement of the maximum light absorption. For accurate analysis, the sample must be diluted, remeasured and the operator must mathematically compensate for the dilution during the calculation of analyte concentration.
Measurement in a colorimeter where the samples are in a flowing stream generates a continuum of absorbance data with respect to time. The response of the system to a sample is a smooth "peak" where the response moves away from a "baseline" or constant light absorption, goes through a maximum and subsequently returns to baseline after the sample has passed through the colorimeter. The gradation of absorbance in a peak is created by dilution occurring at the interface between the sample and the carrier in the flowing stream. The shape of the peak remains constant for a given system, but peak height and area of the peak change proportionally to concentration of analyte in a sample.
Traditional methods of calculating concentration are based on peak height or peak area used in a ratiometric relationship given by the equation: ##EQU1## Both area and peak height measurements require that the method of detection always accurately measure the highest absorbance; that the detection circuitry be linear in response; and that a high enough signal to noise ratio be present to measure very low concentrations. The restraint for these parameters prevents the accurate measurement of analyte concentration over wide ranges and requires that dilution of samples which have concentrations of the analyte which absorb outside of the dynamic range of the detection system be carried out prior to analysis. This nullifies many of the advantages of automated instrumentation. In spite of this, peak area and peak height are the commonly used measurements for calculating the concentration of an analyte in colorimetric chemistry.
A third parameter, peak width is also mathematically correct for calculating concentration (peak area is proportional to peak height X peak width) and in theory could be used, but in practice this has not been possible by direct measurement. The difficulty with this approach is the imprecision of measuring the width of a normally generated peak due to the asymptotic nature of the curve at the baseline.
This invention overcomes previous problems associated with apparatus and methods for determining analyte concentrations in solutions. No longer is a very sophisticated colorimeter required. The invention does not need to be concerned with linearity of electronic response to changes in light energy and low signal to noise ratios at low light levels. Moreover, the invention is accurate over a wide range of concentrations including very high sample concentrations when normally a reliable value could not be determined since the detected signal would be outside the operating range of the colorimeter.
The invention uses the general principles of flow injection analysis (FIA). However, a stirred dilution chamber is introduced into the system which significantly alters the operation and performance of the system. When such a device is introduced into an FIA system, the time sample concentration profile observed at the detector is modified considerably compared to normally generated peak shapes. As the sample, that is confined to a small volume relative to the volume of the stirred dilution chamber, first enters the chamber, the concentration of analyte in the chamber increases dramatically. Subsequently, the level of analyte goes through a maximum concentration and then decreases following an exponential function created by the continuous dilution of sample by addition of newly entering reagent. Of significance to this invention is that as concentration of analyte in the sample increases, the time required to "clear" the sample from the dilution chamber also increases. In this case, time is proportional to the logarithm of the sample concentration.
The advantage of such a device and relationship in an analytical system is that only time from appearance to disappearance of the sample at the detector needs to be measured. Concentrations of analyte in unknown samples can then be calculated from standard curves which establish the time sample concentration relationship represented by the following equation: ##EQU2## This relationship has been proven to hold true for concentrations over ranges of concentration not possible in conventional colorimetric, phosphorescent or fluorescent methods.
Now, since only the appearance or disappearance of analyte exiting from the dilution chamber must be detected, for those analytes with inherent light absorption characteristics or which are coupled to a colored, phosphorescent or fluorescent indicator, a simplified detector can be used. This detector can comprise an appropriate light emitting diode (LED), a flow cell or section of instrument tubing through which the colored analyte is observed, a photosensitive diode detector and associated electronics. In operation, the electrical signal from the output of the photodiode is monitored. As the colored analyte appears in the flow cell between the LED and photodiode detector the intensity of the electrical signal changes. This signal continues to change until the analyte and sample have cleared the dilution chamber and detector. At a predetermined level above the baseline as measured by the photodiode detector (trigger level) a timer is started. The timer is stopped when the signal from the photodiode detector returns to the trigger level. The resulting measured time is proportional to the concentration of analyte in the sample. As a result, wide ranges of concentrations of analyte in unknown samples can be determined without regard to previous instrumental problems which limited the accurate measure of parameters from which concentrations are calculated.
This invention is applicable to any analyte which absorbs light or that can be associated with a colored complex. Examples of such analytes include ammonia, hydrogen ions, phosphate, a large number of inorganic ions including nickel, thallium, iron and many other inorganic and organic compounds important in society and commerce. In addition, analytes that are fluorescent or luminescent or that can be coupled to a fluorescent or luminescent molecule can also be analyzed with this invention. In this case the detector must be changed to measure the appearance and disappearance of fluorescence or luminescence rather than color. In addition, analytes that emit nuclear radiation can also be measured.